Human complement C3 derivates with cobra venom factor-like function

ABSTRACT

A modified human complement C3 protein (C3) is disclosed comprising a substitution of a portion of a human C3 protein, with a corresponding portion of a Cobra Venom Factor protein (CVF) which results in a human C3 protein with CVF functions, but with substantially reduced immunogenicity. Advantageously, the C3 protein can be manipulated to contain at least one of the following CVF functions: increased stability of the C3 convertase and increased resistance to the actions of factors H and/or I. A large number of hybrid C3 proteins containing substitutions in the C-terminal portion of the alpha chain of C3 are presented and tested for the above functions. Methods of treatment of diseases such as reperfusion injury, autoimmune diseases, and other diseases of increased complement activation are presented as well as methods of increasing the effectiveness of gene therapeutics and other therapeutics.

RELATED APPLICATIONS

This International application claims priority to U.S. Provisional Applications 60/567,069, filed Apr. 30, 2004, 60/653,247, filed Feb. 14, 2005, and 60/667,352, filed Mar. 30, 2005, each of which is incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates generally to chimeric derivatives of Human Complement C3 having a substitution of a portion of a human C3 protein with a corresponding portion of a Cobra Venom Factor (CVF) protein. Preferably, a portion of the alpha chain of C3 is substituted with the corresponding portion of CVF.

BACKGROUND OF THE INVENTION

The third component of complement, C3, plays a pivotal role in both the classical and alternative pathways of complement activation, and many of the physiologic C3 activation products have important functions in the immune response and host defense. In the alternative pathway, the activated form of C3, C3b, is a structural subunit of the C3 convertase. This bimolecular enzyme consists of C3b and Bb, the activated form of factor B. This enzyme is formed by the binding of C3b to factor B that is subsequently cleaved by factor D, resulting in the formation of the C3 convertase, C3b,Bb, and the release of the activation peptide Ba. The C3 convertase activates C3 by cleaving the molecule into C3b and the anaphylatoxin, C3a. The C3b molecule will bind to a cell or particle in close proximity to the C3 convertase. Eventually, the bound C3b will allow for the activation of C5 into C5b and the anaphylatoxin, C5a. C5 activation occurs by the same C3b,Bb enzyme that can cleave C5 when it is bound to an additional C3b molecule to produce a trimolecular complex composed of (C3b)₂,Bb. This C5-cleaving trimolecular enzyme is called C5 convertase. Inasmuch as the activation of both C3 and C5 occurs at the identical active site in the Bb subunit, the enzyme is also called C3/C5 convertase; and only one EC number has been assigned (EC 3.4.21.47).

Cobra venom contains a structural and functional analog of C3 called cobra venom factor (CVF). This molecule can bind factor B in human and mammalian serum to form the complex, CVF,B, which is also cleaved by factor D into the bimolecular enzyme CVF,Bb and Ba. The bimolecular complex CVF,Bb is a C3/C5 convertase that activates C3 and C5 analogously to the C3/C5 convertase formed with C3b. Although the two C3/C5 convertases, C3b,Bb and CVF,Bb, share the same molecular architecture, the active site-bearing Bb subunit, and the substrate specificity, the two enzymes exhibit significant functional differences. The CVF,Bb enzyme is physiochemically far more stable than C3b,Bb, it is resistant to inactivation by the regulatory proteins factors H and I, it exhibits different kinetic properties, and it does not require additional C3b for C5 cleavage.

CVF and mammalian C3 have been shown to exhibit several structural similarities including immunologic cross-reactivity, amino acid composition, circular dichroism spectra, secondary structure, electron microscopic ultrastructure, and amino acid sequence. Nevertheless, significant structural differences exist between the two molecules. Whereas C3 is a two-chain molecule with an apparent molecular mass, dependent on the species, of 170 to 190 kDa, CVF is a three-chain molecule with an apparent molecular mass of 149 kDa that resembles C3c, one of the physiologic activation products of C3. Another significant structural difference between C3 and CVF lies in their glycosylation: CVF has a 7.4% (w/w) carbohydrate content consisting mainly of N-linked complex-type chains with unusual α-galactosyl residues at the non-reducing termini. In contrast, human and rat C3 exhibit a lower extent of glycosylation with different structures of their oligosaccharide chains.

Whereas CVF,Bb and C3b,Bb are both C3/C5 convertases, they exhibit important differences. The CVF-containing enzyme is far more stable than the C3-containing enzyme. Both convertases will spontaneously decay into their two respective subunits. However, the intrinsic half-life (stability) of the CVF-containing convertase is approximately 7 hours at 37° C., several hundred times longer than the C3-containing enzyme with an intrinsic half-life of approximately 1.5 minutes. Furthermore, the CVF-containing enzyme as well as free CVF are not subject to regulation by the complement regulatory proteins factors H and I. The combination of the long intrinsic half-life and the resistance to regulation of the CVF-containing enzymes allows CVF to continuously activate C3 and C5 (and subsequently other complement components), ultimately resulting in depletion of the serum complement activity.

Based on the involvement of the complement system in multiple diseases, including diseases of major prevalence, the last decade has seen the development of multiple anti-complementary agents to interfere with the unwanted complement activation process in these disease states. All complement-oriented drug development attempts are based on inhibiting the activation of complement, while CVF acts by depleting complement in serum. Of interest for the treatment of diseases of complement activation is a C3-type molecule which combines the non- or low immunogenicity of C3, with the complement-depleting function of CVF.

SUMMARY OF THE INVENTION

The following listing of embodiments is a nonlimiting statement of various aspects of the invention. Other aspects and variations will be evident in light of the entire disclosure.

Some embodiments include one or more modified human complement C3 proteins, which can have a substitution of a portion of a human C3 protein, with a corresponding portion of a Cobra Venom Factor protein of a sequence substantially related thereto. In some embodiments the substituted portion of the CVF can be within the alpha chain of C3. In other embodiments, the substituted portion of the CVF can be a C-terminal portion of the alpha chain of C3. In some embodiments, the substituted C-terminal portion can include amino acid 1663 of the human C3 protein. In some embodiments, the substituted C-terminal portion can be an internal portion that does not extend through the entire C-terminus of the human C3 protein. In further embodiments, the modified protein can have substantially the same number of amino acid residues as an unmodified human C3 protein. In some embodiments, the substitution can include any positions within amino acid positions 700-1663 of the human C3 protein. Other embodiments are human complement C3 proteins, which can have a substitution of a portion of a human C3 protein, with a corresponding portion of a Cobra Venom Factor protein of a sequence substantially related thereto and which can have at least two substitutions. In some embodiments, the substitution has a selected beginning position and a selected last position; in some such embodiments, the beginning position can be, for example, 749, 874, 936, 994, 1264, 1348, 1496, 1504, 1550, and the like; the last position can be, for example, 784, 921, 970 1324, 1550, 1617, 1663, and the like. In preferred embodiments, the one or more substitutions can include any of amino acids: 1550-1663, 1504-1663, 1348-1663, 1550-1617, 1504-1617, 1496-1663, 1348-1617, 1496-1617, 1264-1324, 749-784, 874-921, 994-1663, 994-1550 and 936-970. In some embodiments, the substituted portion of CVF can be within the beta chain of C3.

In some embodiments, the modified C3 protein can have an affinity for factor B and can support formation of a convertase. In some embodiments, the resulting convertase can cleave C3 and not C5. In further embodiments, the convertase can have an intrinsic half-life between about 1.5 minutes and about 7 hours at 37° C. In some embodiments, the resulting convertase can have an intrinsic half-life of at least about 7 hours at 37° C.

In some embodiments, the modified C3 protein can be expressed as a single chain protein. In some embodiments, the modified C3 protein can be cleaved into at least two chains in a form that resembles C3. In further embodiments, the modified C3 protein can be cleaved to release a C3a portion therefrom. In some embodiments, the modified protein can have an additional 1 to about 19 amino acids at the N-terminus that are not encoded by C3 or CVF. In some embodiments, the modified protein can include a non-C3 signal peptide, such as a Drosophila Bip signal sequence. In some embodiments, the modified C3 protein can have modified affinity for factor B and/or factor D. In some embodiments, the modified protein can show partial or complete resistance to Factor H and/or Factor I. In some embodiments, the modified C3 protein can be substantially non-immunogenic.

Other embodiments can include a method for depleting complement by administering a modified C3 protein to a patient in an amount effective for the depletion of complement. In some embodiments, the administration can be local. In further embodiments, the local administration can be into an organ, subcutaneously, into a cavity, or into a tissue. In other embodiments, the local administration can employ a targeting function capable of concentrating the modified C3 protein in a desired location. In further embodiments, the targeting function can include using an antibody conjugated to the modified C3 protein. In some embodiments, the administration can be a systemic administration, such as intravenous or intraperitoneal.

Further embodiments can be methods for avoiding or ameliorating reperfusion injury in a patient by delivering a modified C3 protein to the patient, sufficient to deplete complement; and permitting reperfusion in the patient. In some embodiments, the delivering step can include injecting the modified C3 protein into an artery. In other embodiments, the delivering step can include a local delivery of the modified C3 protein. In other embodiments, the delivering step can include a systemic delivery of the modified C3 protein. In some embodiments, reperfusion can include opening a blocked artery. In some embodiments, the reperfusion can occur in connection with transplantation of an organ.

Some embodiments can include methods for increasing the efficiency and/or effectiveness of gene therapy by delivering a modified C3 protein in an amount sufficient to deplete complement, providing the gene therapy; and observing an enhanced result therefrom.

Some embodiments can include methods of increasing delivery of a therapeutic or diagnostic agent by delivering a modified C3 protein sufficient to increase blood flow; and providing the therapeutic or diagnostic agent. In some embodiments, the method can include chemically linking the modified C3 protein to an antibody with an affinity for a specific tissue prior to the delivering step. In some embodiments, the antibody can be attached to the modified C3 by recombinant DNA technology. In some embodiments the antibody can be a monoclonal antibody.

Some embodiments include methods of treating an autoimmune disease, comprising administering the modified C3 protein sufficient to deplete complement. In some embodiments, the administration can be episodic and corresponds to periods of at least one elevated disease symptom. In some embodiments, the autoimmune disease can be any of asthma, systemic lupus erythematosus, glomerulonephritis, rheumatoid arthritis, Alzheimer's disease, multiple sclerosis, myocardial ischemia, reperfusion, sepsis, hyperacute rejection, transplant rejection, cardiopulmonary bypass, myocardial infarction, angioplasty, nephritis, dermatomyositis, pemphigoid, spinal cord injury and Parkinson's disease.

Some embodiments include methods of mimicking the properties of CVF in a human protein, by, for example, substituting a portion of a human complement C3 protein with the corresponding portion of a Cobra Venom Factor (CVF) protein. In some embodiments the portion can be within the alpha chain of C3. In other embodiments, the portion can be a C-terminal portion of the alpha chain of C3. In some embodiments, the C-terminal portion can include amino acid 1663 of the human C3 protein. In some embodiments, the substituted C-terminal portion can be an internal portion that does not extend through the entire C-terminus of the human C3 protein.

Other embodiments include methods of selecting a modified C3 protein, by characterizing at least one property of the modified C3 protein to form a function profile of the modified protein; and matching the function profile with a disease or condition to be treated. In some embodiments, at least one property can be selected from the group consisting of: convertase activity, convertase formation, convertase stability, susceptibility to Factor H, susceptibility to Factor 1, ability to cleave C3, and ability to cleave C5. In some embodiments, the selected C3 protein participates in formation of a convertase adapted for treatment of a chronic condition. In some embodiments, the adaptation can include any of, for example, long plasma half-life, high stability, resistance to Factor H, resistance to Factor I, and the like. In some embodiments, the convertase can be adapted for treatment of a reperfusion injury. In other embodiments, the adaptation can be any of, for example, high convertase activity, resistance to Factor H, resistance to Factor I and the like.

Some embodiments include a nucleic acid sequence encoding a modified C3 protein, and/or a vector including the nucleic acid and/or a host cell containing the vector. In some embodiments, the host cell can be any of: a Drosophila S2 cell, an Sf9 cell, a CHO cell, a COS-7 cell, a HiFive cell, a yeast cell, a BHK cell, an HEK293 cell, and an E. coli cell.

Some embodiments include a composition that can include the modified human complement C3 protein and a pharmaceutically acceptable carrier and/or the nucleic acid.

Some embodiments include an expression system expressing the modified C3 protein. In some embodiment, the expression system include a cell selected from the group consisting of: a Drosophila S2 cell, an Sf9 cell, a CHO cell, a COS-7 cell, a HiFive cell, a yeast cell, a BHK cell, an HEK293 cell, and an E. coli cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the chain structures of C3 and CVF with shaded portions present in the mature proteins.

FIG. 2 shows a map of the original CVF/cobra C3 hybrid proteins, showing the region of CVF that was substituted with cobra C3 sequences in each of the five hybrid proteins.

FIGS. 3A-3G show the cDNA and derived amino acid sequence of CVF1 (SEQ ID NOs:3 and 4). The NH₂— and C-termini of the α-, γ-, and β-chains, functionally important regions, and known ligand binding sites are indicated. Amino acid residue numbering starts at the NH₂-terminus of the pro-CVF1 molecule;

FIG. 4 shows results of a complement depletion assay in human serum of the three modified human C3 proteins (HC3-1348, HC3-1504, and HC3-1550) as compared to CVF.

FIG. 5 shows results of an assay to measure the ability of the modified human C3 proteins (HC3-1348, HC3-1504, and HC3-1550) to activate factor B and to form a C3/C5 convertase as compared to CVF and C3b.

FIG. 6 shows results of an assay to measure the activity of C3/C5 convertase to activate C3 for the three modified human C3 proteins (HC3-1348, HC3-1504, and HC3-1550) as compared to CVF.

FIG. 7 shows results of an assay to measure C3 cleavage (using 20% of the amount of convertase used in the experiment in FIG. 6) of the modified human C3 protein HC3-1348 as compared to CVF.

FIG. 8 is a summary of the results for the activity measurements of the three modified human C3 proteins: HC3-1348, HC3-1504, and HC3-1550 as compared to CVF and Human C3b.

FIG. 9 is a graph showing the results of complement depletion of Natural CVF and a variety of human C3 proteins.

FIG. 10 is a graph showing the results of Cleavage of factor B by C3b, Recombinant CVF and a variety of modified human C3 proteins.

FIG. 11 is a graph showing the results of C3 cleavage of Natural CVF and a variety of modified human C3 proteins. The inset graph is of the same reaction performed using 20% of the convertase in the main graph.

FIG. 12 is a graph showing the results of cleavage of selected hybrid proteins by factors H and I.

FIG. 13 is a picture of a gel showing the results of C5 conversion by Human C3/CVF hybrid proteins.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The replacement of human Complement C3 sequences with CVF sequences representing key structural requirements for CVF specific functions allows the creation of C3 derivatives with CVF-like functions. Preferred embodiments of the invention provide human C3 derivatives that exhibit the CVF-specific function of depleting complement by forming a stable convertase for use as a novel therapeutic agent to deplete complement in clinical situations where complement activation is part of the pathogenesis. Because the structural changes in human C3 caused by CVF-specific sequences are minimal, the modified C3 molecule will exhibit significantly reduced or even absent immunogenicity.

A number of modified human complement C3 proteins (C3) are disclosed that have a substitution of a portion of a human C3 protein, with a corresponding portion of a Cobra Venom Factor protein (CVF) which results in a human C3 protein with CVF functions, but with substantially reduced immunogenicity. The C3 protein may be the pro-protein (a single chain protein) or the cleaved protein by removal of four arginine residues. An additional two chain form may be formed by the removal of the C3a portion. Advantageously, the C3 protein can be manipulated to contain at least one of the following CVF functions: increased ability to form the C3 convertase, increased stability of the C3 convertase, increased resistance to the actions of factors H and/or I, increased activity to cleave C3 and C5, and increased plasma half-life. In one embodiment, the C-terminal portion of the alpha chain of C3 is substituted with a corresponding portion of a CVF protein. A wide variety of specific regions and specific chimerae are disclosed herein. Further, a method of identifying and/or selecting a modified C3 protein is disclosed which involves characterizing the properties of the modified C3 protein to form a function profile of the modified protein, and matching the function profile with a disease or condition to be treated. The function profile may include one or more of the following functions: ability to form a convertase, susceptibility to H and/or I, the ability to cleave C3, the ability to cleave C5, the relative activity of the convertase, the stability of the convertase, and the plasma half-life. The modified C3 protein, also referred to as a chimeric C3 protein, a C3/CVF chimera, or a C3/CVF hybrid protein, can be used to treat a variety of diseases and/or conditions that result from local or systemic complement activation. These diseases include, but are not limited to: reperfusion injury, autoimmune diseases such as rheumatoid arthritis and lupus, and myasthenia gravis and other diseases that result from antibodies that recognize and direct an immune reaction against proteins or structures of the human body.

The Complement System and Disease

The complement system is a component of the vertebrate immune system which is involved in maintaining health through its roles in host defense and immune response. However, complement activation is also involved in the pathogenesis of a multitude of diseases. Examples of these diseases include autoimmune hemolytic anemias, rheumatoid arthritis and other immune complex diseases, and reperfusion injuries in which tissue damage occurs after blood flow has been temporarily suspended. Examples of reperfusion injuries are tissue damage after the reopening of blocked vessels (e.g. heart attack, stroke) and reperfusion with a recipient's blood after organ transplantation. Based on the involvement of the complement system in multiple diseases, including diseases of major prevalence, the last decade has seen the development of multiple anti-complementary agents to interfere with the unwanted complement activation process in these disease states. All of these drug development attempts are based on inhibiting the activation of complement.

Complement Protein C3, Cobra Venom Factor CVF, Similarities and Differences

The third component of complement, C3, plays a pivotal role in both the classical and alternative pathways of complement activation, and many of the physiological C3 activation products have important functions in the immune response and host defense (for review see Müller-Eberhard, H. J. (1988) “Molecular Organization and Function of the Complement System,” Ann. Rev. Biochem., 57:321-347, herein incorporated by reference in its entirety). Human C3 is a two-chain glycoprotein with a molecular weight of approximately 185,000. It is synthesized as single-chain pre-pro-C3 which undergoes subsequent processing by removal of four arginine residues between the β- and α-chains. The primary sequence of human C3 is known from molecular cloning. Full or partial sequence information of C3 from other mammalian species as well as non-mammalian species is available including mouse, rat, guinea pig, chicken, cobra, Xenopus, and lamprey. The human C3 gene is 42 kb in length and includes 41 exons, ranging in size from 52 to 213 bp.

The major activation product of C3 is C3b. C3b plays a central role in the formation of the alternative pathway C3/C5 convertase. The formation of this enzyme requires initial binding of C3b to Factor B. The weak complex C3b,B is subsequently cleaved by Factor D in the presence of Mg²⁺, resulting in the enzymatically active C3/C5 convertase C3b,Bb and in release of the activation peptide Ba. The C3b,Bb enzyme is very labile, exhibiting spontaneous decay-dissociation into the two subunits C3b and Bb with an intrinsic half life of 1.5 minutes at 37° C. The C3b,Bb enzyme is stabilized by properdin. The C3/C5 convertase cleaves C3 and C5 by hydrolyzing a single peptide bond in the α-chains of the two substrates. In order for the enzyme to cleave C5, C5 has to be bound to another C3b molecule bound to the convertase. In addition to the fast spontaneous decay-dissociation, the C3b,Bb enzyme is subject to stringent control. The enzyme is disassembled by Factor H, and C3b is inactivated by the combined action of factors H and I. In the presence of Factor H, Factor I cleaves the α′-chain of C3b at two cleavage sites. The resulting C3b derivative, called iC3b, can no longer form a convertase with Factor B. Factor I can cleave the α′-chain at a third site, which causes the generation of the two C3 fragments C3c and C3dg. For the third cleavage by Factor I, the C3b receptor, CR1 serves as co-factor.

An unusual structural property of C3 is the presence of an intramolecular thioester in the α-chain. Upon activation of C3 to C3b, the thioester becomes highly reactive and is responsible for the covalent attachment of C3b to cellular and other particular targets. The structural change which accompanies cleavage of the thioester allows the subsequent binding of Factor B and its activation.

The thioester in C3 undergoes slow spontaneous hydrolysis, resulting in the formation of a form of C3 called iC3 or C3(H₂O). iC3 assumes C3b-like functions and can form a fluid-phase convertase with Factors B and D in serum. The resulting convertase iC3,Bb is similarly labile as the C3b,Bb convertase and subject to control by factors H and I. However, spontaneous hydrolysis of the thioester and the ensuing low grade activation of C3 by the iC3,Bb convertase is believed to be responsible for the initial deposition of C3b on target cells or particles, leading to activation of the alternative pathway on so-called activator surfaces.

C3 is a highly unusual multi-functional protein. The protein including its various activation products specifically interacts with approximately twenty different plasma proteins or cell surface receptors. This multifunctionality has spurred significant interest in a detailed structure/function analysis of the molecule. For some ligands of C3, including Factor H, properdin, Factor B, and the complement receptors CR1, CR2, CR3, and C3a receptor binding sites have been proposed or assigned to more or less defined regions of the C3 polypeptide.

Cobra venom contains a structural and functional analog of C3 called cobra venom factor (CVF). Functionally, CVF resembles C3b in that it can bind Factor B in human serum and virtually all vertebrate sera to form a weak complex CVF,B, which is subsequently cleaved by Factor D in the presence of Mg²⁺ into the bimolecular enzyme CVF,Bb and Ba. The bimolecular complex CVF,Bb is a C3/C5 convertase that activates C3 and C5 analogously to the C3/C5 convertase formed with C3b.

CVF is a three-chain glycoprotein with a molecular mass of approximately 150,000 Da. CVF and mammalian C3 have been shown to exhibit several structural similarities including immunological cross-reactivity, amino acid composition, circular dichroism spectra and secondary structure, and electron microscopic ultrastructure. Initial N-terminal amino acid sequence comparisons have demonstrated sequence homology with C3 and have led to the suggestion that CVF structurally resembles C3c. The structural homology between CVF and C3 and the chain relationships were confirmed by the molecular cloning of CVF, which revealed an overall similarity at the protein level of approximately 70 percent to mammalian C3s and over 90 percent when compared to cobra C3.

Despite these functional and structural similarities between CVF and C3, the two molecules and the resulting convertases exhibit important functional differences:

-   -   1. Both enzymes exhibit spontaneous decay-dissociation into the         respective subunits which abolishes the enzymatic activity.         Whereas the C3b,Bb enzyme is very short-lived and decays with an         intrinsic half-life of 1.5 minutes at 37° C., the CVF,Bb enzyme         is orders of magnitude more stable, decaying with an intrinsic         half-life of approximately seven hours.     -   2. The C3b,Bb enzyme is subject to regulation by factors H         and I. In contrast, CVF,Bb and CVF are completely resistant to         the regulatory actions of these two proteins.     -   3. The C3b,Bb enzyme generated during complement activation is         surface bound. In contrast, the CVF,Bb enzyme is a fluid-phase         enzyme (like iC3,Bb).     -   4. Another functional difference between C3b,Bb and CVF,Bb lies         in the C5 convertase activities. In order for C5 to be cleaved         by a C5 convertase, it needs to be bound to either C3b or CVF.         However, for C5 cleavage to occur by the C3b,Bb enzyme, C5 has         to be bound to a different C3b molecule than the one that is         part of the C3b,Bb enzyme. In contrast, C5 is bound by the same         CVF molecule that carries the Bb catalytic subunit. This         property of the CVF,Bb enzyme to bind C5 is probably the reason         for its ability to exhibit fluid phase C5 convertase activity,         whereas the C5 convertase activity of the C3b,Bb enzyme is         confined to the surface of a particle.     -   5. Both enzymes have been shown to differ somewhat in their         kinetics of C3 hydrolysis. Based on the k_(cat)/K_(m), the         catalytic efficiency is approximately eight-fold greater for         C3b,Bb compared to CVF,Bb.

In terms of functional consequences, the two most significant differences between CVF,Bb and C3b,Bb are the intrinsic stability of the CVF,Bb enzyme and its resistance to the regulatory proteins factors H and I. Once the CVF,Bb enzyme has formed, it will continue to activate C3 and C5, leading to complement consumption. Ever since it was demonstrated over 30 years ago that CVF can be administered safely to laboratory animals in order to deplete their plasma complement, CVF has become an important investigational tool to study the various biological functions of complement in immune response, host defense, and pathogenesis of disease by comparing normal (complement-sufficient) animals with CVF-treated (complement-depleted) animals.

C3/CVF Derivatives.

CVF is a complement inhibitor that acts through a mechanism of exhaustive activation which subsequently leads to depletion. As a matter of fact, CVF is frequently used as the standard to evaluate the anti-complement activity of other drugs. Whereas CVF exhibits this powerful anti-complement activity, it is not suitable for human application because of its immunogenicity. For this reason it is desirable to prepare a substantially non-immunogenic CVF by taking advantage of the extensive structural similarity between CVF and C3, and to generate human C3 derivatives with the desired complement-depleting function of CVF by substituting the functionally important regions of the CVF molecule into human C3 by recombinant means. This has been accomplished as described herein.

A number of human C3 derivatives have been produced and/or designed, in which portions of the C3 sequence were replaced with homologous CVF sequences, mainly sequences in the CVF beta chain. CVF and C3 have been successfully expressed by recombinant means in eukaryotic expression systems. The alpha chain region of C3, and preferably the C-terminal portion of this chain, was chosen based on previous work in which five hybrid proteins were constructed between CVF and cobra C3 (because of its greater similarity to CVF than human C3), collectively spanning the entire CVF sequence (Mol. Immunol. 40:199 (2003), incorporated herein by reference in its entirety). The other line of work involved the limited proteolysis of the CVF protein (Mol. Immunol. 30, Suppl. 1, 113 (1993) U.S. Pat. No. 5,174,344, incorporated herein by reference in its entirety). Using the results of this previous work, three human C3 preferred derivatives were created by replacing amino acid residues 1550-1663, 1504-1663, and 1348-1663, respectively, with the homologous sequences from CVF. The C3 derivatives are designated by the first amino acid residue and if the last amino acid residue is not indicated, it is understood to be 1663. If the endpoint of the CVF insertion is before the C-terminus of the protein, it is designated with the location (for example: HC3-1550/1617). The C3 derivative which was created by replacing amino acids residues 1348-1663 was formerly referred to as 1325-1663, but substitution mapping has subsequently shown that the substitution was at 1348 rather than 1325. The three human C3 derivatives are referred to as HC3-1550, HC3-1504, and HC3-1348. The three human C3 derivatives were shown to exhibit the desired CVF activity: all three proteins are able to form an active C3/C5 convertase. This is demonstrated by the ability of the three proteins to support the activation of factor B, and by the ability of the three resulting convertases to cleave C3. All three proteins form stable convertases, although the intrinsic stability and at least one (HC3-1550) exhibits a lower intrinsic stability compared to CVF. Unexpected was the observation that two of the three proteins first tested (HC3-1550 and HC3-1348) were actually able to decomplement guinea pig serum, although this activity was clearly less than that of CVF, and four of five proteins were able to deplete complement in human serum, with HC3-1348 and HC3-1496 able to decomplement human sera almost as well as CVF. HC3-1550/1617 was not able to deplete complement in human serum. The ability to at least partially deplete complement indicates that the C3 derivative does not only form a stable convertase but is at least partially resistant to the regulatory proteins factors H and/or 1, thereby exhibiting CVF-like activity. The human C3 derivative HC3-1550 differs from human C3 in less than 4% of the amino acid residues, which greatly reduces or eliminates its predicted immunogenicity compared to CVF. Various C3 hybrids can be preferred, based upon their various characteristics, depending upon the disease to be treated. For example, for the treatment of chronic diseases, the objective is to provide a C3 hybrid having extremely low or no immunogenicity and high convertase stability, facilitating its persistence in the body of the patient suffering from the chronic disease. In such a situation, the other characteristics, such as the activity of the convertase, are of comparatively less importance. In contrast, for treatment to avoid complement-associated reperfusion injury, a high convertase activity is of greater importance than convertase stability. Thus, a multiplicity of different hybrids, each having a particular array of properties, permits selection of a preferred hybrid for treatment of a given condition that can be treated by complement activation and/or complement depletion.

In one embodiment, the present invention relates to modified complement C3 proteins that exhibit at least one of the following CVF or CVF,Bb qualities: decaying with an intrinsic half-life of longer than 1.5 minutes, increased resistance to the regulatory actions of factors H and/or I, fluid-phase C3 convertase and fluid-phase C5 convertase activity. In addition to these factors, in some embodiments the catalytic efficiency may be reduced relative to C3b,Bb, since the catalytic efficiency is approximately eight-fold greater for C3b,Bb compared to CVF,Bb. In other embodiments the catalytic efficiency is not reduced or is elevated in comparison to C3b,Bb. Although many preferred C3 hybrids have little or no immunogenicity, other embodiments, which are nevertheless suitable for many applications, may display detectable to moderate immunogenicity in some cases.

In some embodiments, the intrinsic half-life of the convertase formed with the modified C3 protein is greater than 1.5 minutes, preferably greater than 10 minutes. In further embodiments, the intrinsic half-life can fall generally between that of the CVF-containing convertase (7 hours or longer) and that of C3 (1.5 minutes), including but not limited to about: 2 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours and 7 hours, or more. Modified C3 proteins with short convertase intrinsic half-lives and/or short plasma half-lives will be useful for some applications, while C3 proteins with long convertase intrinsic half-lives and/or long plasma half-lives will be useful for different applications.

In further embodiments, the resistance of the C3 hybrid to factors H and/or I is greater than that for the unmodified C3 and is in some embodiments as good as the resistance of CVF. However, in some embodiments, further modification in other parts of the molecule can be necessary to achieve resistance to factors H and/or 1.

While many embodiments described herein are directed to specific substitutions of one or more discrete regions of C3 with corresponding regions of CVF, other embodiments include substitutions employing a sequence that is substantially related, but not identical, to a CVF sequence. That is, there are positions within a CVF region selected for substitution, in which changes to one or more amino acids can be made without a loss of any desirable feature or function of the selected CVF region, and in some cases such changes can confer an enhanced feature or function. All such changes are considered to be embodiments of the invention.

In a further embodiment, the catalytic activity of convertase containing the modified C3 protein is in some embodiments at least 50% that of the convertase containing CVF, and may be greater than that of the convertase containing unmodified C3. In a further embodiment, the catalytic activity is 60%, 70%, 80% 90% or 100% that of the CVF convertase. Both enzymes have been shown to differ somewhat in their kinetics of C3 hydrolysis. Thus, in many embodiments, convertases containing the modified C3 can have a catalytic activity that falls between the two, or that exceeds the activity of the convertase containing unmodified C3. Thus, in some embodiments, such activity of the convertase containing the modified C3 can be from 10% to 1000%, or more, that of the convertase containing CVF, including but not limited to 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 110%, 135%, 150%, 200%, 300%, 400%, 500%, 750%, 1000% and more.

Based on the k_(cat)/K_(m), the catalytic efficiency is approximately eight-fold greater for C3b,Bb compared to CVF,Bb when cleaving C3. Thus, in some embodiments, the catalytic efficiency of convertase containing the modified C3 protein is in some embodiments at least 50% that of the convertase containing CVF, and may be greater than that of the convertase containing unmodified C3b. Both enzymes have been shown to differ somewhat in their kinetics of C3 hydrolysis. Thus, in many embodiments, convertases containing the modified C3 can have a catalytic efficiency that falls between the two, or that exceeds the efficiency of the convertase containing unmodified C3. Thus, in some embodiments, such efficiency of the convertase containing the modified C3 can be from 10% to 1000%, or more, that of the convertase containing CVF, including but not limited to 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 110%, 135%, 150%, 200%, 300%, 400%, 500%, 750%, 1000% and more.

In further embodiments, the C5 cleaving activity of the modified C3 proteins is increased. The C5 cleaving activity can be from about 10% to 400% of the activity of CVF or C3, including but not limited to: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, and 375%.

In further embodiments, the binding of the modified C3 proteins to Factor B and/or its subsequent cleavage by factor D may be reduced. However, as long as at least a functional amount of the catalytic activity and the complement depleting activity remain, the modified C3 protein is useful.

In further embodiments, convertases having the modified C3 proteins exhibit substantially the same complement-activating activity of those containing natural CVF. By the term “exhibit substantially the same complement-activating activity of natural CVF it is meant that the C3 derivatives of the present invention have from 0.1 to 97%, preferably from 50 to 97%, preferable from 80 to 97% of the level of the complement activating activity of natural CVF as measured by the method of Cochran et al. ((1970) J. Immunol. 105(1)), 55-69, herein incorporated by reference in its entirety).

In some embodiments, the modified complement C3 proteins are C3 molecules in which some or all of the C-terminal region has been replaced with the corresponding region of CVF. In some embodiments, only the C-terminal portion of the alpha chain of C3 is replaced with the corresponding region of CVF. In some embodiments some or all of amino acids 700-1663 are replaced, including but not limited to regions of from 20 to about 1000 amino acids, including but not limited to: 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 175, 180, 190, 200, 250, 275, 300, 350, 375, 400, 450, 475, 500, 550, 575, 600, 650, 675, 700, 750, 775, 800, 850, 875, 900, 950 975, 1000. In some embodiments, the beta chain of C3 is intact, that is, is the same as in natural C3. However, in some embodiments, in addition to the substitutions in the alpha chain of C3, other substitutions can be made to provide such functions as increased stability (for example, the sites which relate to Factor H binding and Factor I cleavage can be mutated or substituted). Specific substitutions include but are not limited to amino acids 1550-1663, 1504-1663, 1348-1663, 1550-1617, 1504-1617, 1470-1663, 1348-1617, 1470-1617, 1264-1324, 1348-1386, 749-784, 874-921, 1496-1663, 1496-1617 and 936-970. In some embodiments, still smaller substitutions in these areas can correspond to smaller regions that result in a C3 with the desired CVF functions or qualities.

The immunogenicity of preferred embodiments remains low or absent—comparable to that of C3. In some embodiments, the modified C3 protein is substantially non-immunogenic. Substantially non-immunogenic means that the protein can still exhibit complement activating function when injected into a human patient. Further, the modified C3 protein may be as non-immunogenic as C3 or may be from about 75% non-immunogenic to about 100% non-immunogenic, including but not limited to 80%, 85%, 90%, 95%, and 99%.

Methods of Treatment Systemically and Locally

The modified human complement C3 proteins produced by the methods disclosed herein can be used to deplete complement locally or systemically as follows:

Local treatment may be effected in a number of ways to produce a result of depletion of complement or activation of complement, depending on the desired effect. In one embodiment, local depletion is effected when the modified C3 proteins are administered locally to an organ, tissue, cavity, or intradermally. This results in a temporary and complete depletion of complement in the area. Local depletion or activation may also be effected using an insulin-type pump that produces an intermittent or constant flow of the modified C3 protein to a selected site. Alternatively, local activation of complement may employ a specific monoclonal antibody which, when chemically attached to the modified C3, can localize it to a specific tissue, a disease, or an infected cell to cause continuous activation of complement in that area. In other embodiments, the antibody can be attached to the modified C3 protein via recombinant DNA technology.

Systemic depletion is effected when the modified C3 proteins are administered systemically, for example, intravenously or intraperitoneally. This results in a temporary and complete depletion of complement systemically. This method can be used for reperfusion injury, coronary heart surgery, transplantation and or systemic disease, particularly during a flare-up or episodical activity. This is further discussed in the Examples provided herein.

Many of the most advantageous qualities of the modified human C3 proteins used in each of these cases and in specific disease states can vary considerably. For example, in those cases when an immediate, but temporary removal of complement is desired, a modified protein which has a shorter plasma half-life and/or stability, but high complement activation activity (high C3/C5 convertase activity) would be most suited. In the case where a chronic disease is being treated, a long plasma half-life and/or stability and a low or sluggish activity would be most suitable.

Convertase Activities/Functions

When producing and analyzing the best use for the modified C3 proteins herein, a number of functions and activities can be used for such an analysis, including but not limited to the following:

Affinity for Factor B—Differences in how efficiently the modified C3 proteins support the activation of factor B (cleavage into Ba and Bb) can be detected and taken advantage of. There are multiple factors beyond just affinity for factor B that go into supporting factor B activation, but once the convertase is formed, the subsequent key properties that are generally most important are stability, regulation by H and I, and activity of cleaving C3 and C5.

Stability—Both the C3b,Bb and CVF,Bb enzymes exhibit spontaneous decay-dissociation into the respective subunits which abolishes the enzymatic activity. Whereas the C3b,Bb enzyme is very short-lived and decays with an intrinsic half-life of 1.5 minutes at 37° C., the CVF,Bb enzyme is orders of magnitude more stable, decaying with an intrinsic half-life of approximately seven hours.

Regulation by Factors H and/or I. The C3b,Bb enzyme is subject to regulation by factors H and I. In contrast, CVF,Bb and CVF are completely resistant to the regulatory actions of these two proteins.

Activity of Cleaving C3—Both enzymes have been shown to differ somewhat in their kinetics of C3 hydrolysis. Based on the k_(cat)/K_(m), the catalytic efficiency is approximately eight-fold greater for C3b,Bb compared to CVF,Bb.

Immunogenicity—Because the structural changes in human C3 caused by CVF-specific sequences are minimal, modified C3 can exhibit significantly reduced or even absent immunogenicity as compared with CVF. In addition, as is typical in species far removed from human, a transferase enzyme is produced in cobras that transfers an alpha-bonded galactose to the end of CVF oligosaccharide chains. Because humans do not produce this transferase, the alpha bonded galactose residues are seen as non-self and are highly immunogenic. Thus, anti-alpha-gal antibodies are produced that recognize the specific CHO moieties produced on CVF. Human C3 and CVF lack identity at 52% of their positions, and carbohydrate differences are even more pronounced. Thus, because preferred modified C3 proteins have less than 10%, and in some embodiments about 4%, of the C3 amino acids replaced with those of CVF, and also have no cobra-derived carbohydrate structures (specifically alpha-bonded galactoses on the glycans), the C3 hybrids of the invention are significantly less immunogenic. In some embodiments, the C3 hybrids of the preferred embodiments are significantly less immunogenic than CVF. In other embodiments, the C3 hybrids of the preferred embodiments are at least 50% less immunogenic than CVF. In some embodiments, the recombinant proteins are produced in either insect or mammalian cell lines, which will result in carbohydrate moieties that are either simple or very similar to what would be found in humans.

EXAMPLES

Five CVF/cobra C3 loss-of-function hybrid proteins were produced in which large portions of the CVF sequence were replaced by homologous portions of cobra C3. Preliminary characterization of these hybrid proteins showed that substitutions in the α-chain of CVF (β-chain of C3) with cobra C3 sequences did not appear to change the functional property of depleting serum complement activity, whereas replacing portions of the β- and γ-chains had a major effect. Hybrid proteins H4 and H5, in which CVF residues 978 through 1642 were replaced by cobra C3 sequence, exhibited a significant reduction in bystander lysis (a measure for C5 cleavage) activity in addition to the impaired ability to deplete serum complement. This suggested that the alpha chain of C3 (corresponding to the β- and β-chains of CVF) may be a major site for the differences between the activity of CVF and C3. Another line of experimental support strongly suggests the C-terminal portion of the CVF β-chain is important for CVF function comes from the experiments of limited proteolysis of CVF with chymotrypsin (Grunwald, et al. (1993) Mol. Immunol. 30, Supp. 1, 30, herein incorporated by reference in its entirety). Thus, a large number of modified human complement C3 proteins (C3) have been produce or identified that have a substitution of a portion of the human C3 protein, with a corresponding portion of the Cobra Venom Factor protein (CVF). These substitutions result in a human C3 protein with CVF-type functions, but with substantially less immunogenicity than CVF. The production and testing of these proteins is set forth below in Examples 1-3. Various uses and assays for these proteins are provided in Examples 4-10.

Example 1 Production of Human Complement C3/CVF Hybrid Proteins

The replacement of human C3 sequences with CVF sequences representing important structural features for CVF specific functions allows the creation of C3 derivatives with CVF-like functions. Thus, embodiments of the invention are directed to generation of human C3 derivatives that exhibit the CVF-specific function of depleting complement by forming a stable convertase for use as a novel therapeutic agent to deplete complement in clinical situations where complement activation is part of the pathogenesis. Because the structural changes in human C3 caused by CVF-specific sequences are minimal, it is clear that the modified C3 molecules can exhibit significantly reduced or event absent immunogenicity.

The human C3 molecules in Table 1 are engineered to contain specific CVF sequences so as to create human C3 derivatives with CVF functions. Some embodiments of the invention provide smaller substitutions in these areas, to define small regions that result in modified C3 proteins that can form a relatively stable convertase and that exhibit little or no immunogenicity.

TABLE 1 Exemplary Human C3/CVF clones HC3-1550 HC3-1504 HC3-1348 HC3-1550/1617 HC3-1504/1617 HC3-1470 HC3-1348/1617 HC3-1470/1617 HC3-1264/1324 HC3-1348/1386 HC3-749/784 HC3-874/921 HC3-936/970 HC3-1496 HC3-1496/1617 HC3-994 HC3-994/1617 HC3-1617

Certain modified C3 proteins (also called hybrid proteins or chimerae) were produced by site-directed mutagenesis as described below. Briefly, for site-directed mutagenesis replacing small portions of human C3 sequence with CVF, the procedure of Ho et al. was used (Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. and Pease, L. R. (1989) “Site-Directed Mutagenesis by Overlap Extension Using the Polymerase Chain Reaction” Gene, 77:51-59, herein incorporated by reference in its entirety). In this method, two PCR reactions were performed, one with a forward primer somewhere upstream from the site of the desired mutation. The second, reverse primer contained the mutation. The second PCR of this round had a forward primer containing the desired mutation, with the reverse primer being downstream from the mutation site. At least one unique restriction site is preferably present in each of the PCR products from this step so that it is possible to transfer the modified DNA back into the original clone. Amplification was done using a large amount of template DNA, and a low number of cycles to minimize mutations introduced by the PCR process. More specifically, the first round of PCR, the reaction for the 5′ product used a human C3 plasmid as the template, while the other PCR used a CVF plasmid. In this case the middle, “mutagenesis” primers partially consisted of C3 sequences and partially of CVF sequences, providing a “bridge” between the two sequences.

After amplification, the two products were purified by gel electrophoresis and isolated from the gel using a Qiaquick Gel extraction kit from Qiagen. Then, the fragments were combined, and another PCR reaction done, using the two fragments as the template, and the outside primers as the amplification primers. Again, the PCR reaction was performed using a high concentration of template DNA and only cycled for a few cycles to minimize PCR-caused mutations. The resulting PCR product was cut with the two unique restriction enzymes, size purified on an agarose gel, and the fragment of interest isolated using the Qiaquick column. The fragments were then cut with the appropriate enzymes, and cloned into either pBS-HuC3 or pHC3-1550(-sig) that had been cut with the same enzymes.

The first hybrid plasmid, pHC3-1550 contained CVF sequences replacing the homologous C3 sequences from position 1550 to the C-terminus of the protein. The second hybrid plasmid, pHC3-1504, codes for a hybrid protein containing CVF sequences replacing human C3 sequences from position 1504 to the C-terminus of the protein. The third hybrid plasmid, pHC3-1348, contained CVF sequences replacing the homologous C3 sequences from position 1348 to the C terminus of the protein. To prepare the first plasmid, two initial PCR reactions were performed. Both hybrids had about 19 amino acid vector-coded sequence having some or all of the following RSPWPGVPTSPVWWNSADA (SEQ ID NO:5). Because of the method of cloning of the C3 gene, these amino acids coded for by the cloning vector, were N-terminal to the N-terminus of human C3. These extra amino acids did not affect the activity of the protein, so they could be removed with no ill effect. The first plasmid was prepared using pBS-HC3-2 as a template, and using the following oligonucleotides as primers: HC3H5-1 (GGATGCCACTATGTCTATATTGGACATATCC—SEQ ID NO:6), and HC3H5-2 (TCTTCTATTCGAACCAGTCGGGTCTTGTAC—SEQ ID NO:7). The second PCR used pCVF-FL3Δ as a template, and the following oligonucleotides as primers: HuC3H5-3 (GTACAAGACCCGACTGGTTCGAATAGAAGAACAAG—SEQ ID NO:8) and HuC3H5-4 (TATCATGTAAGCGGCCGCGTATAAACAATTTAAGGG—SEQ ID NO:9). Both reactions were performed in an Eppendorf thermocycler, using the following program: 95° C. for 5 min., followed by five cycles of 95° C. for 30 sec., 58° C. for 30 sec. (gradient from 50 to 65° C.), and 72° C. for 1 min., followed by 20 cycles of 95° C. for 30 sec., 57.5° C. for 30 sec. (gradient from 55 to 60° C.), 72° C. for 1 min, followed by 72° C. for 10 min. The two fragments were purified using a Qiagen PCR cleanup kit, and joined in a second PCR reaction, using HC3H5-1 and HC3H5-2 as primers, and the two primary PCR products as template. The cycling conditions used for this reaction were: 95° C. for 5 min, followed by 5 cycles of; 95° C. for 30 sec., 51° C. for 30 sec (gradient from 46 to 56° C.), and 72° C. for 1.5 min, followed by 20 cycles of; 95° C. for 30 sec., 57° C. for 30 sec (Gradient from 52 to 62° C.), and 72° C. for 1.5 min. This was followed by a 10 min incubation at 72° C. The PCR fragment was purified as described above, cut with BsrGI and NotI, gel purified, and isolated using a Qiagen Gel Isolation kit. This fragment was cloned into pBS-HuC3-2 that had been cut with the same enzymes, and the resulting clones were screened for insertion of the correct fragment by digestion with EcoRI. All clones with the correct EcoRI digestion pattern were sequenced to ascertain that no PCR-induced mutations were inserted. A large-scale preparation of one clone (pHC3-1550) with the expected sequence was performed, and the insert excised by digestion with HindIII (and end repaired with T4 DNA polymerase) and NotI. The fragment was purified by gel electrophoresis, isolated from the gel as described above, and cloned into the Drosophila expression vector, pMT/V5-HisA that had been cut with EcoRV and NotI. Attempts to obtain expression of hybrid proteins from this construct resulted in very low yields of protein. For this reason, a construct was made having the human C3 signal sequence removed from pHC3-1550, while inserting a new, unique AfeI site. To do this, pHC3-1550 was amplified with the following two primers: HC3SigRemF; AGATCTCCATGGAAGCTTAGCGCTGGGAGTCCCATGTACTCTATCATC (SEQ ID NO:10, and HC3SigRemR: GCGTCCCGCCTfCAACAGCC (SEQ ID NO:11). After amplification, the fragment was purified as described above, cut with HindIII and SpeI. The 150 bp band was gel isolated, and cloned into pHC3-1550 that had been cut with the same enzymes. DNA from transformants was screened by cutting with AfeI, and all positive clones were confirmed by DNA sequencing. This plasmid was called pHC3-1550(-sig).

The insert was excised from the plasmid by digestion with AfeI, DraI (to fragment the plasmid), and NotI. The digest was run on a gel, and the 5 kb fragment isolated as described above. It was then ligated into pMT-Bip/V5-HisA that had been digested with EcoRV and NotI. The resulting plasmid was called pMB/HC3-1550.

The plasmid for the production of the second hybrid protein, HC3-1504, was produced in a similar manner, as follows. Two PCR reactions were performed to obtain the human C3 and CVF portions of the coding sequence. In the first, pBS-HuC3-2 was used as a template, with the following oligonucleotides used as primers: HC3H5-3-F1(TCTGTGTGGCAGACCCCTTCGAGG—SEQ ID NO:12) and HC3H5-3—R1 (CGTrACCAATACATATCTTGTTCAGCTTTCCATCC—SEQ ID NO:13). The second PCR used pCVF-FL3Δ as a template, and the following oligonucleotides as primers: HuCC3H5-3-F2 (GGATGGAAAGCTGAACAAGATATGTATTGGTAACG—SEQ ID NO:14), and HuC3H5-3-R2 (CATCCATGACATAGATATCATTACCATCTTG—SEQ ID NO:15). The resulting two PCR products were joined in a PCR reaction, using HuC3H5-3-F1 and HuC3H5-3-R2 as primers, and the two PCR fragments as the template. After the second PCR reaction, the product was purified using a Qiagen PCR cleanup kit, it was then cut with NspV, and cloned into pHC3-1550(-sig) that had been cut with the same enzyme and that had also been treated with Calf intestine alkaline phosphate. Resulting clones were cut with EcoRI to determine the orientation of the insert and sequenced to ascertain that no PCR induced modifications were present. The resulting plasmid was called pHC3-1504. The insert from this plasmid was then isolated as described above, and cloned into pMT-Bip/V5-HisA as described above. This plasmid was called pMB/HC3-1504.

The plasmid for the production of the third construct, HC3-1348, was constructed in a similar manner to that used for HC3-1504. The only difference is that the two mutation primers were HuC3H5-5-1R (GCAACTGTGCGTTATACATTGTCACCACCGAC—SEQ ID NO:16) and HuC3H5-5-2F (GTCGGTGGTGACAATGTATAACGCACAGTTGC—SEQ ID NO:17). For the primary PCR reactions, the primers used were HuC3H5-3-1F and HuC3H5-5-1R, and the template was pBS-HuC3-2, while the primers used for the second primary PCR reaction were HuC3H5-5-2F and HuC3H5-3-2R, using pCVF-FL3Δ as the template. After the primary PCR, the two fragments were purified and used as templates for the secondary PCR reaction as described for the construction of pHC3-1504. The secondary PCR reaction product was purified, cut with NspV, and cloned into pHC3-1550, the sequence confirmed and the insert cloned into pMT-Bip/V5-HisA) as described above.

The plasmid for the production of the fourth hybrid protein, HC3-1496, was produced in a similar manner, as follows. Two PCR reactions were performed to obtain the human C3 and CVF portions of the coding sequence. In the first, pBS-HuC3-2 was used as a template, with the following oligonucleotides used as primers: HC3H5-3-F1(TCTGTGTGGCAGACCCCTTCGAGG—SEQ ID NO:12) and HC3H5-4-R1 GAGAAGGCCTGTTCCTTTATCCGGATGGTAGAACCGGGTAC (SEQ ID NO:18) and. The second PCR used pCVF-FL3Δ as a template, and the following oligonucleotides as primers: HuCC3H5-4-F2 CCGGTTCTACCATCCGGATAAAGGAACAGGCCTTC (SEQ ID NO:19), and HuC3H5-3-R2 (CATCCATGACATAGATATCATTACCATCTTG—SEQ ID NO:20). The resulting two PCR products were joined in a PCR reaction, using HuC3H5-3-F1 and HuC3H5-3-R2 as primers, and the two PCR fragments as the template. After the second PCR reaction, the product was purified using a Qiagen PCR cleanup kit. It was then cut with NspV, and cloned into pHC3-1550(-sig) that had been cut with the same enzyme and been Calf intestine alkaline phosphate treated. The resulting plasmid was called pHC3-1496. The insert from this plasmid was then isolated as described above, and cloned into pMT-Bip/V5-HisA as described above. This plasmid was called pMB/HC3-1496.

The plasmid for the production of the fifth hybrid protein, HC3-1550/1617, in which the C-terminal 46 amino acid residues of HC3-1550 are replaced with human C3 sequences, is described below. Again, two PCR reactions were done to obtain the CVF and human C3 portions of the coding sequence. In the first, pHC3-1550 was amplified, using the following two primers; HuC3H5-F1 GGATGCCACTATGTCTATATTGGACATATCC (SEQ ID NO:21), and HuC3H5-2R1, CCCGATGATGTAGCTGAGTTTATCTTTCGTGGG (SEQ ID NO:22). The second PCR was performed using pCVF-FL3Δ as the template, and HuC3H5-2F2 (CCCACGAAAGATAAACTCAGCTACATCATCGGG—SEQ ID NO:23) and HuC3H5-2-R2 (AATTGGAGCTCCACCGCGGTGG—SEQ ID NO:24) as the primers. After the first PCR, the fragments were joined in a second PCR reaction, using HuC3H5-F1 and HuC3H5-2-R2 as the primers, and the two PCR fragments as the template. Following this PCR, the amplified fragment was purified using Qiagen PCR purification columns, cut with BsrGI and NotI, and cloned into pHC3-1550(-sig) that had been cut with the same enzymes. The resulting plasmid was sequenced to ascertain the correct sequence. It was called pHC3-1550/1617. The insert was isolated as described above and cloned into pMT-Bip/V5-HisA as described above. This plasmid was called pMB/HC3-1550/1617.

In some cases, portions of the human C3 are additionally exchanged with CVF sequence at more than one site to generate a modified C3 that is useful for one or more of the purposes presented herein. Thus, modified C3 proteins are generated wherein CVF-specific sequence is inserted in more than one region, or known regions are mutagenized by various means. For example, in addition to a site or sites required for the formation of a physically stable convertase with factor B, it can also be desirable to change a Factor I cleavage site in human C3. Factor I-resistant mutants of human C3 have been successfully described previously by Fecke, et al., 1998 (Fecke, W., Farries, T. C., D'Cruz, L. G., Napper, C. M., and Harrison, R. A. (1998) Xenotransplantation 5:29-34, herein incorporated by reference in its entirety). For example, replacement of select sequences required for specific CVF functions allows one to engineer novel C3 derivatives wherein specific subsets of CVF functions are present or eliminated, respectively (e.g. a C3 derivative or CVF derivative that forms a stable C3 convertase but does not cleave C5). A modified C3 molecule that does not activate C5 may have particular advantage for therapy as it prevents the generation of the pro-inflammatory C5a anaphylatoxin.

Example 2 Expression of Modified human C3 Proteins

The proteins were produced in the Drosophila S2 cell system, using the Drosophila Bip signal sequence for secretion of the proteins. Briefly, the plasmids pMB/HC3-1550, pMB/HC3-1504, pMB/HC3-1496, pMB/HC3-1550/1617, and pMB/HC3-1348 were transfected into Drosophila S2 cells using the calcium phosphate method of Chen and Okayama (Chen, C., and Okayama, H. (1987) Mol. Cell. Biol. 7(8), 2745-2752, herein incorporated by reference in its entirety). S2 cells were transfected with a mixture of expression plasmid and pCoBlast, using a ratio of 19:1 (w:w). Following transfection, cells containing both plasmids were selected using blasticidin (25 μg/ml). For expression, 1-liter cultures of transfected cells were grown in serum-free medium (Hi-Five plus L-glutamine), in the absence of blasticidin. When the cells reached a density of 5×10⁶ cells/ml., production of the recombinant proteins was induced by the addition of CuSO₄ to a final concentration of 25 μM. Cultures were allowed to express recombinant proteins for 4-5 days. Hybrid proteins were then purified from the media by a combination of ANX, Sephacryl H-300, and CM-FF chromatography.

Because of the method of cloning of the C3 gene, there are several amino acids (approximately 19) coded for by the cloning vector, that are N-terminal to the N-terminus of human C3. These extra amino acids do not affect the activity of the protein, so they can be removed with no ill effect. In some cases, it can be preferred for there to be at least two amino acids that are an artifact of the restriction sites needed to clone the final construct into the expression vector. In various embodiments, a variety of signal sequences can be used, including the native signal sequence of human C3, as well as any other signal sequence effective in directing entry of the nascent polypeptide into the endoplasmic reticulum.

Other expression systems that can be used, include but are not limited to: Baculovirus infection of Sf9 or HiFive cells (other insect expression systems), CHO cells, COS-7 cells (mammalian expression systems), E. coli, BHK, HEK293 cells, and various yeast expression systems, including the Hanselula yeast expression system.

Example 3 Results of the Activity Measurements of the Modified Human Complement C3 Proteins

The purified modified human C3 protein hybrids were subjected to a number of functional analyses as follows.

Complement Depletion:

This assay measures the ability of a protein to deplete complement in human (or other) serum. The assay was done in two steps. In the first step, the protein of interest was diluted to the desired concentrations in buffer, usually by serial dilution (typically from less than a nanogram/microliter up to approximately 320 ng/microliter or 3.2 μg in the 10 microliters used in the assay). Then, a 10 μl aliquot of the diluted protein was mixed with undiluted serum. The mixture was incubated at 37° C. for 3 hours, which allows the protein to activate complement by forming a C3 convertase. The convertases formed were then able to activate C3 in the serum. Then, to measure the amount of complement activity left, the serum was diluted and mixed with antibody-sensitized sheep erythrocytes, which are easily lysed by complement when it is present in serum. This reaction was allowed to proceed for 30 minutes, and was stopped by diluting the mixture in cold buffer. The cells were centrifuged, and the lysed cells quantified by measuring the hemoglobin released. The results are shown in FIG. 4 and FIG. 9.

As expected, very small amounts of CVF were able to completely deplete the complement in human serum. 800 ng of either of the proteins HC3-1348 and HC3-1496 was able to completely deplete 10 μl of human serum. Other hybrid proteins were less active, requiring about 34 μg of protein to partially deplete 10 μl of human serum of complement. One hybrid protein, HC3-1550/1617 was apparently unable to deplete complement at the concentrations examined.

Unexpected was the observation that two of the proteins (HC3-1550 and HC3-1348) were actually able to deplete complement in guinea pig serum, although this activity was clearly less than that of CVF. Notably, preferred embodiments HC3-1550, HC3-1504, HC3-1496 and HC3-1348 were all able to deplete complement in human serum.

Factor B Activation Assay.

This was an assay to measure the ability of a hybrid protein to activate factor B, and form a C3/C5 convertase. The convertase formation was measured as a function of the cleavage of factor B into Bb and Ba. In the assay, purified hybrid proteins were incubated with a three-fold excess of factor B and factor D (all highly purified) in the presence of magnesium at 37° C. At various times, aliquots of the reaction were withdrawn, and the reaction stopped by adding EDTA, which chelates the magnesium. The reaction products were run on a non-reducing SDS-polyacrylamide gel, which was stained for proteins with Coomassie Blue. The amount of Factor B converted was quantified by scanning the gel into a specialized computer program and measuring the amount of protein in the factor B and Bb bands.

The results in FIGS. 5 and 10 show that Factor B was activated very rapidly in the presence of Human C3, for two reasons. Human C3 was able to bind factor B very rapidly, which makes it available for cleavage by factor D. However, the resulting convertase was very unstable, and falls apart rapidly, making the C3b available to bind more factor B. The reaction in the presence of CVF was much slower, which was a result of the lower affinity of CVF for factor B, and the greater stability of the CVF containing convertase. The conversion of factor B in the presence of HC3-1504 or HC3-1496 was quite similar to that of CVF. HC3-1550 and HC3-1550/1617 were able to convert factor B much more rapidly than CVF, but slower than C3b. This was probably a result of the convertase being less stable than the CVF containing enzyme, but more stable than the C3b containing convertase. In addition, it is likely that the initial binding of factor B by HC3-1550 is much more rapid than by CVF. Finally, HC3-1348 supports the cleavage of factor B less well than the other proteins discussed. This is probably a combination of the resulting convertase being more stable than the C3b containing enzyme, and the initial binding of factor B being less rapid.

C3 Convertase Activity Assay

This assay measures the activity of C3/C5 convertases containing hybrid proteins to activate human C3, by cleaving off the C3a peptide. To perform this assay, convertases were formed as described above, and the reaction stopped by the addition of EDTA. The convertase was then mixed with human C3, and the reaction incubated at 37° C. At the indicated times, aliquots were removed, and the reaction stopped by mixing with gel loading buffer containing SDS and β-mercaptoethanol. The SDS denatures the proteins, and the β-mercaptoethanol reduces the disulfide bonds between cysteines in the proteins. After electrophoresis under reducing conditions, the gel was stained with Coomassie Blue dye, and the relative amounts of the C3 α-chain and C3 α′-chain quantified as described above. Care was taken to use the same amount of convertase in each reaction.

FIG. 6 and FIG. 11 show that in this assay, CVF and HC3-1550 both were able to convert human C3 at approximately equal rates. HC3-1504 was markedly slower than CVF, but still was able to completely convert the amount of C3 present within one hour. HC3-1348 and HC3-1496 appeared to convert C3 at a rate faster than CVF, while HC3-1550/1617 appeared to convert C3 at an initially high rate which became slower after about 10 minutes. To further investigate this phenomenon, the C3 conversion assay was repeated, using all proteins except HC3-1504. In an effort to reduce the speed of the reaction, the amount of convertase present was reduced by a factor of five. These results are shown in FIG. 7 and in the insert of FIG. 11. In this assay, it was clear that the HC3-1348 and HC3-1496 formed convertases that were markedly efficient than CVF. HC3-1550/1617 formed a convertase that was initially active, but appears to become mostly inactive after about 10 minutes.

C5 Conversion Assay

The assay for C5 conversion activity was done essentially as described by Petrella et al., (1987) J. Immunol. Methods 104(1-2), 159-172, herein incorporated by reference in its entirety. In this assay, C5 convertase was formed as described above, using a total of 3 μg protein. After convertase formation, the reaction was stopped by the addition of EDTA to a final concentration of 5 mM. Then, 5 μl of this reaction was added to a 25 μl reaction containing 7 μg C5 in PBS. The reaction was incubated at 37° C. for 24 hours, and the reaction stopped by the addition of 7 μl Laemmli gel loading buffer, followed by boiling for 5 minutes. The reaction products were separated by reducing SDS-PAGE, and the gel was stained with Coomassie Blue dye, and the relative amounts of the C5 α-chain and C5 α′-chain quantified as described above. None of the current proteins are able to form an active C5 convertase.

The assay for degradation of the proteins by factors H and I was performed essentially according to the method of Oran and Isenman ((1999) J. Biol. Chem. 274 (8), 5120-5130, herein incorporated by reference in its entirety). In this method, 12 μg of each protein was incubated with 4.3 μg factor H and 0.3 μg factor I at 37° C. in a total volume of 60 μl. At the indicated times, 10 μl aliquots were withdrawn, and reactions stopped by the addition of 5 μl 5× Laemli gel loading buffer. Reaction products were separated by SDS-PAGE on a 4-20% gradient gel under reducing conditions. These data show that all proteins are partially resistant to digestion by factor I in the presence of factor H. In a similar assay, C3b was nearly digested to completion at the 0 timepoint.

Example 4 A Method for Depleting Complement Locally or Systemically

The modified human Complement C3 proteins produced by the methods disclosed herein are used to deplete complement locally or systemically as follows:

Local depletion is effected when the modified C3 proteins are administered locally to an organ, tissue, cavity, or intradermally. This results in a temporary and complete depletion of complement in the area. Alternatively, local depletion may use a specific monoclonal antibody which, when chemically attached to the modified C3, would localize it to a specific tissue, a disease site, or an infected cell to cause continuous depletion of complement in that area.

Systemic depletion is effected when the modified C3 proteins are administered systemically, for example, intravenously or intraperitoneally. This results in a temporary and complete depletion of complement systemically. This method can be used for reperfusion injury, coronary heart surgery, transplantation and/or systemic disease, particularly during a flare-up of symptoms or during episodical activity.

Some of the most advantageous qualities of the modified human C3 proteins used in each of these cases and in specific disease states can vary considerably. For example, in those cases for which an immediate, but temporary depletion of complement is desired, a modified protein having a shorter plasma half-life and/or lower stability, but high complement activation activity is preferred. In treating a chronic disease, a long plasma half-life and/or high stability, even if accompanied by a low or sluggish activity, would be preferred. Further, a modified C3 molecule that does not activate C5 can be particularly advantageous for certain therapies as it prevents the generation of the pro-inflammatory C5a anaphylatoxin.

Example 5 A Method for the Treatment of Reperfusion Injury

Examples of reperfusion injuries are tissue damage after the reopening of blocked vessels (e.g. after a heart attack or ischemic stroke), and reperfusion of a transplanted organ with a recipients' blood. For a clogged coronary artery or for reperfusion after organ transplantation, it can be desirable in many cases to deplete the complement before the transplant is reperfused or before the opening of the blocked vessel. The ability to avoid complement activation can avoid the tissue damage it causes, so the only remaining primary source of tissue damage is oxygen starvation. Typically, tissue damage due to complement activation during reperfusion is twice as great as tissue damage due to oxygen starvation—that is, roughly ⅔ of tissue damage is attributable to complement activation while ⅓ is attributable to oxygen starvation. Thus, reperfusion injury can be greatly diminished by depleting complement prior to reperfusion, as is possible with embodiments of the present invention. In this case it is preferable to use the highest activity convertase and to use a high dosage. The stability of the convertase is less important than it would be for a chronic disease.

In general terms, the methods of these embodiments of the invention involve administering an effective amount of a modified C3 protein systemically allowing enough time for depletion of complement, and then performing the surgery.

Example 6 A Method for Increasing the Effectiveness and/or Efficiency of Gene Therapy

This method relies on the depletion of complement in order to help prolong survival of a useful virus in the body. Because complement has been found to help the removal from the body of certain viral vectors used in gene therapy, it is desirable to reduce the amount of circulating complement prior to administration of a gene therapy vector. This may be done locally or systemically depending upon the type of gene therapy that is being used.

The method involves administering an effective amount of a modified C3 protein systemically or to the local area where the gene therapy is being administered, allowing time for depletion of complement, and then administering the gene therapy.

Example 7 A Method for Increasing Delivery of a Therapeutic (e.g. Chemotherapeutic) or Diagnostic Agent

To increase the blood flow to an area where a therapeutic is being administered, a modified C3 protein is chemically linked to a monoclonal antibody with an affinity for the tissue of choice. In this example, the modified C3 is one that forms a highly active C3/C5 convertase. The modified C3/antibody is targeted to the tissue, resulting in local complement activation in the region, causing vessel permeability due to complement activation. The vessel permeability continues as long as the active convertase/antibody complexes are bound to the target, because new, non-activated complement is continually supplied by the blood arriving at the target.

For use of this method during the treatment of lung cancer, a modified C3 protein-monoclonal antibody hybrid is administered to a lung cancer patient, using a monoclonal antibody that recognizes a lung-specific antigen. The antibody binds to lung tissue and activates complement locally. This increases the vessel permeability in the lung, permitting the chemotherapeutic agent to more efficiently and effectively act on the lung cancer. As described above, this method permits continuous local activation of complement, which permits a persistent local elevation in blood-vessel permeability.

Example 8 A Method of Treating Rheumatoid Arthritis, Lupus and Other Autoimmune or Immune Complex Diseases

This method uses the example of rheumatoid arthritis as one of several conditions in which pain and inflammation arise from complement activation in a local area. For treatment, a modified C3 protein is administered systemically or locally, diminishing the complement response/activation by depleting the complement. This can reduce the symptoms of the disease and the progression of the disease. It can also be beneficial to have episodical depletion in combination with a longer term lowering of the activity of the complement system, especially when there are episodes of exacerbation of the symptoms of the disease. Further, this method can be used with other diseases with circulating immune complexes, such as lupus and other autoimmune diseases.

For example, when a complement-activating autoantibody is produced and is directed against the body's own proteins, the disease effect is primarily due to the binding of the antibody to the target causing complement activation and tissue damage as well as other interference with normal function. Myasthenia Gravis is an example of this type of disease. Autoantibody binds to the neuromuscular endplate where the nerve comes into contact with the muscle. Complement is activated and blocks the neurotransmitter, resulting in paralysis. Employing the method of this embodiment of the invention, the systemic depletion of complement, either continuously or during exacerbations in the disease, can markedly reduce symptoms and progression thereof.

Example 9 Method of Selecting a Modified C3 Protein

Various modified C3 proteins are useful for different diseases, and methods of treatment. Thus, it is useful to analyze the functional qualities of the modified C3 proteins of embodiments of the invention and to use them accordingly. The following methods are employed to analyze the function of purified modified C3 proteins produced as in Example 2. The methods described herein, as well as others that are known to those of skill in the art, may be used.

Assays to determine convertase activity. In addition to the specific assays as mentioned below, two hemolytic assays for depletion of serum complement activity and induction of bystander lysis can be employed for screening.

Complement depletion assay. To measure the anticomplementary (complement consumption) activity of modified C3 proteins, a small volume of human serum is incubated with CVF or hybrid proteins for three hours or shorter periods of time at 37° C. at a protein concentration of 5 μg/ml, to allow the proteins to deplete complement. The remaining complement hemolytic activity is subsequently measured using sensitized sheep erythrocytes using methods known to one of skill in the art including that of Cochrane et al., 1970 (Cochrane et al., 1970, J. Immunol. 117:630-4, herein incorporated by reference in its entirety).

Bystander lysis assay. The bystander lysis assay is performed by incubating 20 μl of normal guinea pig serum at 37° C. with 20 μl of CVF or hybrid proteins at a concentration of 5 μg/ml and 20 μl guinea pig erythrocytes (5×10⁸/ml). The CVF or hybrid proteins participate in fluid-phase activation of C5, which leads to lysis of the erythrocytes. Thus, presence of hemoglobin in the supernatant is indicative of C5 activation. The reaction is incubated at 37° C. for 30 minutes, and is stopped by the addition of 1 ml of cold buffer. After centrifugation, the released hemoglobin is measured spectrophotometrically. (Vogel, C. W., and Müller-Eberhard, H. J. (1984) J. Immunol. Methods 73(1), 203-220, herein incorporated by reference in its entirety).

C3 convertase formation/Factor B activation. To detect cleavage of Factor B into Ba and Bb, a hybrid protein (at 1 μM) is incubated for up to twenty four hours in the presence of a three-fold molar excess of Factor B and 0.5 μM of Factor D in the presence of MgCl₂ at 37° C. The reaction mixtures are analyzed by electrophoresis on 7.5% (w/v) SDS polyacrylamide gels under non-reducing conditions to monitor the disappearance of Factor B and the appearance of the cleavage products Ba and Bb. If necessary, a subsequent western blot can be performed to detect the Ba and Bb cleavage fragments. Controls can include native CVF, pro-CVF, cobra C3, iC3, human C3, iC3, C3b, and EDTA (Vogel and Müller-Eberhard, 1982, J. Biol. Chem. 257:8292-9, herein incorporated by reference in its entirety).

C3 cleaving activity. To examine C3 cleaving activity, a C3 convertase is pre-formed as described herein, in reference to “C3 convertase formation/Factor B activation,” using the hybrid proteins and human Factor B and Factor D. The convertase formation is stopped by the addition of EDTA, and purified human C3 is added The reaction mixture is incubated at 37° C. for one hour or for any other appropriate period of time. Aliquots are taken and immediately transferred into an ice water bath to stop further C3 activation. C3 cleavage is monitored by the disappearance of the C3 α-chain and appearance of the C3 α′-chain by running the reaction products on a 7.5% (w/v) SDS polyacrylamide gel under reducing conditions. If necessary, a subsequent western blot using anti-C3 antiserum is performed. Controls include native CVF, pro-CVF, and human and cobra iC3 or C3b (Vogel and MUller-Eberhard, 1982, J. Biol. Chem. 257:8292-9, herein incorporated by reference in its entirety).

C5 cleaving assay. The C5 cleaving assay is performed as described above for the C3 cleaving assay using purified human C5 as substrate (Petrella et al., 1987, J. Immunol. 164:4742-4751, herein incorporated by reference in its entirety). See, for example, FIG. 12.

Assay for convertase stability. Bimolecular convertases are pre-formed using the hybrid proteins and purified human Factor B and Factor D as described above. After addition of EDTA, the mixture is incubated at 37° C. and aliquots are removed over a period of 24 hours or shorter periods of time if appropriate, and each aliquot is immediately placed in an ice water bath. Subsequently, the C3 convertase activity is determined by the C3 cleaving assay described above. From the reduction of the C3 cleaving activity over time, the half-life of the spontaneous decay-dissociation of the various convertases is calculated. If insufficient quantities of hybrid proteins for this assay are available, the enzymatic activity is determined using the fluorogenic tripeptide t-butyloxy-carbonyl-leucyl-glycyl-arginyl-aminomethylcoumarin. (Caporale, L. H., Gabaer, S. S., Kell, W., and Gotze, O. 1981 J. Immunol. 126(5), 1963-1965, herein incorporated by reference in its entirety).

Assay for Factor H binding. Factor H binding to hybrid proteins is determined using an ELISA assay. Hybrid proteins are adsorbed onto microtiter plates. After blocking with ovalbumin and BSA, purified human Factor H at 10 μg/ml is added and incubated for 30 minutes at room temperature. After washing, bound Factor H is detected with anti-Factor-H antibody followed with an appropriate phosphatase-linked secondary antibody. If factor H can bind to the protein, an appropriate color change is observed. Controls can include native CVF, pro-CVF, cobra and human C3, as well as cobra and human iC3 (Alsenz et al., 1992, Dev. Comp. Immunol. 16:63-76, herein incorporated by reference in its entirety).

Assay for Factor I cleavage. Hybrid proteins are incubated with purified human Factor H and Factor I at 37° C. for several hours. The reactions are analyzed by subsequent 4-20% (w/v) SDS polyacrylamide gel electrophoresis under reducing conditions. Factor I activity is determined by the reduction in the strength of the 105 kDa α′-chain band, and appearance of bands with a molecular weight of 37 and 40 kDa. If necessary, a subsequent western blot is performed using anti-CVF and/or anti-C3 antibodies. Alternatively, hybrid proteins are labeled with ¹²⁵I using the iodogen method (Fraker and Speck, 1978). Cleavage products are detected after SDS polyacrylamide gel electrophoresis by autoradiography (Lambris et al., 1996, J. Immunol. 156:4821-32, herein incorporated by reference in its entirety). See, for example, FIG. 12.

Assays for Immunogenicity. Various methods can be used to analyze immunogenicity, including but not limited to, skin tests, testing the modified C3 protein in transgenic animals which have been genetically engineered to have human immune systems, in vitro methods, including RIA tests using serum generated in such transgenic animals, Radioimmunoprecipitation assays, ELISA assays, Electrochemiluminescence, and Surface Plasmon Resonance. In addition, mouse, rate or guinea pig analogs of some proteins are constructed, using either mouse, rate or guinea pig C3 and CVF sequences. These are injected into the appropriate animal, and serum is collected and analyzed for the production of antibodies against the hybrid proteins.

Example 10 Method of Measuring Plasma Half-Life

There are many factors that can affect the plasma half-life of the modified C3 protein, including but not limited to specific antibodies produced by the immune system, proteases circulating within the serum, non-specific immune responses, and specific regulatory factors such as Factors H and I. In order for the modified C3 proteins to be able to activate and subsequently deplete complement, preferred C3 proteins will persist within human plasma for at least a minimum amount of time. Thus, it is of interest to identify the plasma half-life of the modified C3 proteins to determine how useful they will be for treatment of certain diseases.

This method measures the stability of the modified C3 protein in plasma in three ways. However it is to be understood that one or all of the methods can be used as well as any other methods known to one of skill in the art.

The first method measures the stability in serum in vitro. Human serum is isolated and separated from the whole blood of a patient. Aliquots of different concentrations of the modified C3 proteins are added to the serum and allowed to incubate. Aliquots of the serum are removed at various time intervals and the amount of modified C3 that persists is identified in an ELISA assay using a monoclonal antibody which is specific to C3.

A second method allows for the identification of stability in serum in a humanized animal. The modified C3 protein is administered to the animal and blood samples are taken over time. The amount of modified C3 protein is identified in an ELISA assay using specific antibodies to the protein.

A third method allows for the identification of stability in a human patient. The modified C3 is administered to the patient and blood samples are removed over time. The amount of modified C3 protein is identified using an ELISA assay. This will give a clear indication of how long the modified C3 protein circulates within the plasma of a patient.

In some embodiments, the antibody that is used need not be specific for the modified C3. For example, antibodies that recognize normal C3 can be tested and used to identify the modified C3 in an ELISA procedure.

Example 11 C3 Convertase Formation of Modified Human Complement C3 Proteins as Measured Using Surface Plasmon Resonance

Table 2 shows relative binding of complement proteins to C3, CVF, and recombinant human C3/CVF proteins. In all cases, a higher number indicates that the protein interaction is tighter. The proteins (C3b, etc.) were bound to a BIACORE CHIP™, and then contacted with complement factors. The amount of each of the complement factors bound to the chip (and thus bound to the modified C3 proteins) was determined by surface plasmon resonance. The results showed that: 1) Neither of the recombinant proteins bound C5, consistent with the inability to form a convertase capable of cleaving C5. 2) Both proteins bound factor H, though CVF does not. Factor H is one of the regulatory proteins that is capable of dissociating the C3b,Bb convertase complex and directing a second complement protein, factor I, to inactivate C3b through cleavage. 3) The affinity of the proteins for factor B in the presence of factor D and magnesium was approximately proportional to the rate the proteins were able to form a C3 convertase (as measured by their ability to mediate factor B cleavage—see FIG. 10). Both HC3-1550 and HC3-1348 were cleaved but slower than C3b. CVF is not cleaved at all by H or I, because natural CVF has no H or I sites. It is interesting that recombinant CVF, a 2 chain molecule, has 2 or 3 I sites, but is still not cleaved by factor I.

TABLE 2 Relative binding of Complement proteins to C3, CVF, and recombinant human C3/CVF proteins. Amount of protein bound (RU)-2nd column corrected for MW Protein on Factor B Factor B Factor B Chip C5 Factor H (in EDTA) (with Mg) (with fD and Mg) C3b 607 646 243 285 625 HC3-1550 66 550 9 264 340 HC3-1348 18 219 5 276 162 CVF 617 −14 99 298 370

Table 3 shows the stability of the C3 convertases formed by the modified human Complement C3 proteins as measured by surface plasmon resonance on a BIACORE machine at 25° C. The results showed that both proteins were able to form C3 convertases that were substantially more stable than the C3b-containing convertase. The table also shows that the HC3-1348-containing convertase was actually more stable than the CVF-containing enzyme. These results are consistent with other measurements of half life at higher temperatures which showed that CVF had a half life of 7 hours at 37° C. and C3b had a half life of 1.5 minutes at 37° C.

TABLE 3 C3 convertase formation Protein on Chip T½ of C3 convertase (min) C3b 4.3 HC3-1550 119.3 HC3-1348 1720.0 CVF 1100.0

Example 12 Factor B Cleavage of Modified Human Complement C3 Proteins

The factor B cleavage graph in FIG. 10 shows a time course of the ability of the modified human complement C3b proteins to mediate the cleavage of factor B in the presence of magnesium ion and factor D. This can be used to measure the ability of the protein to form a C3/C5 convertase. Of note in this assay was that C3b formed a convertasc very efficiently, both because it bound factor B very efficiently in the presence of factor D and magnesium (see the data in Example 11), and because the resulting complex was very unstable. CVF, HC3-1496 and HC3-1504 all formed a convertase at approximately the same rate, while HC3-1550 and HC3-1550/1617 were intermediate between C3b and CVF in their ability to form a convertase. HC3-1348 was very slow to bind factor B. This is most probably explained by a combination of a longer half-life of the convertase once formed and a lower affinity of the hybrid protein for factor B.

Example 13 C3 Cleavage of Modified Human Complement C3 Proteins

FIG. 11 shows the ability of the convertases to cleave C3, and shows a time course of the cleavage reaction. The inset is a time course performed with 20% of the convertase used in the main graph. The results showed that CVF and HC3-1550 formed convertases that were approximately equally efficient at C3 cleavage, while HC3-1348 and HC3-1496 both formed convertases that were approximately 5-fold more efficient than CVF at cleaving C3. It is interesting that the HC3-1550/1617-containing convertase appeared to be quite unstable, and did not support C3 cleavage after about 10 minutes.

Example 14 Complement Depletion of Modified Human Complement C3 Proteins

The data in the complement depletion chart in FIG. 9 show that all proteins except HC3-1550/1617 were able to deplete complement, though with very different efficiencies. Both HC3-1550 and HC3-1504 were quite inefficient at the depletion of complement, while HC3-1496 and HC3-1348 were able to deplete complement quite efficiently, though not quite as well as natural or recombinant CVF. HC3-1348 was formerly referred to as HC3-1325 in a previous application, but substitution mapping has subsequently shown that the substitution was at 1348 rather than 1325.

In conclusion, HC3-1496 is an interesting protein because, although it only has an insert 8 amino acids longer than HC3-1504, it acts much more like HC3-1348 with respect to complement depletion and C3 cleavage and more like HC3-1504 with respect to factor B cleavage. It forms a convertase as well as HC3-1504 or CVF, but the resulting convertase is more active at cleaving C3.

HC31550/1617 was made by replacing the region from 1550 to the end of C3 with the CVF region, then taking away the last 46 amino acids and replacing them with C3. This chimeric molecule showed no complement depletion, formed a C3 convertase as well as HC3-1550, and the C3 convertase formed was nearly as active as HC3-550, but had an apparently shorter half-life.

Example 15 Methods of Producing Variants in the Modified C3 Proteins

Variants of the modified C3 proteins are produced that have advantageous qualities. By advantageous it is meant that the variants enhance one or more activities of the protein, including but not limited to: C3 convertase activity, serum decomplementation, factor B binding, cleavage of C3, Binding of Bb and Binding of C3. The variants can include one or more mutations in each region and can include one or more amino acids. Some specific variants are set out below:

Because the C-terminus is involved in stabilization of the C3 convertase, mutations are produced in the C-terminus (after aa 1617) of any modified C3 proteins. The mutations can be insertions, deletions, and substitutions. However, the mutations are preferably substitutions. The mutations stabilize the C3 convertase. Examples of mutations at the C terminus include but are not limited to: mutations at positions 1633, 1654, and 1658. Non-conservative as well as conservative mutations, in addition to amino acid changes that affect the conformation, are included among the embodiments. However, preferably the mutations result in a non-conservative amino acid change.

There are three changes between CVF and human C3 in the span of 1496 and 1504 (8 amino acid residues) that appear to be responsible for a large change in the activity of the convertase. Thus, mutations between positions 1496 and 1504 of any of the modified C3 proteins are included that result in increased activity of the convertase in cleaving C3 and/or in decomplementing serum. The mutations can be changes to one or more amino acids within that region. Preferably, the mutations are substitutions of one or more amino acids in that region, in particular, mutations that result in non-conservative amino acid changes.

Mutations between positions 1348 and 1496 in any modified C3 protein are produced that modify the ability of the protein to bind factor B (and specifically, the ability for C3b and factor B to bind), preferably the modification results in an increased ability to bind to factor B. Whole regions from CVF are switched out to C3 and vice versa. Further mutations are included that result in amino acid substitutions. More specifically, the regions from 1367-1379 are switched for CVF and for C3 and specific amino acids within this region are substituted.

Sequence changes in the region of around 1550 in CVF and 1570-1584 in C3 that may be responsible for the activity of the C3 convertase in cleaving C3, either from binding Bb or binding the target C3 molecule. Using CVF/cobra C3 substitutions, a series of 4 amino acid residues were identified (Q1550G, E1554R, P1556A and R1557Q-positions numbered according to CVF sequence numbering) that resulted in a protein with a much lower activity in cleaving C3. Therefore, variants are produced in the amino acids from position 1570-1584 that result in an increased activity of the C3 convertase in cleaving C3 for the modified C3 proteins. The variants are preferably amino acid substitutions of one or more amino acids in that region.

The various methods and techniques described above provide a number of ways to carry out the invention. Of course, it is to be understood that not necessarily all objectives or advantages described may be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods may be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as may be taught or suggested herein.

Furthermore, the skilled artisan will recognize the interchangeability of various features from different embodiments. Similarly, the various features and steps discussed above, as well as other known equivalents for each such feature or step, can be combined and/or exchanged by one of ordinary skill in this art to perform methods in accordance with principles described herein. Each patent, journal reference, and the like, cited herein is hereby incorporated by reference in its entirety.

Although the invention has been disclosed in the context of certain embodiments and examples, it is understood by those skilled in the art that the invention extends beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and obvious modifications and equivalents thereof. Accordingly, the invention is not intended to be limited by the specific disclosures of preferred embodiments herein. 

1. A modified human complement C3 protein, comprising a substitution of a portion of a human C3 protein, with a corresponding portion of a Cobra Venom Factor protein of a sequence substantially related thereto.
 2. The modified C3 protein of claim 1, wherein the substituted portion of the CVF is within the alpha chain of C3.
 3. The modified C3 protein of claim 2, wherein the substituted portion of the CVF is a C-terminal portion of the alpha chain of C3.
 4. The modified C3 protein of claim 3, wherein the substituted C-terminal portion includes amino acid 1663 of the human C3 protein.
 5. The modified C3 protein of claim 3, wherein the substituted C-terminal portion is an internal portion that does not extend through the entire C-terminus of the human C3 protein.
 6. The modified C3 protein of claim 1, wherein the modified protein has substantially the same number of amino acid residues as an unmodified human C3 protein.
 7. The modified C3 protein of claim 1, wherein the substitution comprises any positions within amino acid positions 700-1663 of the human C3 protein.
 8. The modified C3 protein of claim 1, wherein the substitution comprises a first position and a last position, wherein the first position is selected from the group consisting of 749, 874, 936, ′, 1264, 1348, 1496, 1504, and 1550, and wherein the last position is selected from the group consisting of 784, 921, 970 1324, 1550, 1617, and
 1663. 9. The modified C3 protein of claim 8, wherein the substitution is selected from the group consisting of amino acids: 1550-1663, 1504-1663, 1348-1663, 1550-1617, 1504-1617, 1496-1663, 1348-1617, 1496-1617, 1264-1324, 749-784, 874-921, 994-1663, 994-1550 and 936-970.
 10. The modified C3 protein of claim 1, wherein the modified C3 protein has an affinity for factor B and supports formation of an active convertase.
 11. The modified C3 protein of claim 10, wherein the convertase has an intrinsic half-life of at least about 15 minutes at 37° C.
 12. The modified C3 protein of claim 1, wherein the modified protein has an additional 1 to 19 amino acids at the N-terminus that are not encoded by C3 or CVF.
 13. The modified C3 protein of claim 1, wherein the modified protein is substantially non-immunogenic.
 14. A method for depleting complement, comprising administering the modified C3 protein of claim 1 to a patient in an amount effective for the depletion of complement.
 15. A method for increasing the efficiency and/or effectiveness of gene therapy, comprising: delivering the modified C3 protein of claim 1 in an amount sufficient to deplete complement; providing the gene therapy; and observing an enhanced result therefrom.
 16. A method of increasing delivery of a therapeutic or diagnostic agent, comprising: delivering the modified C3 protein of claim 1 in an amount sufficient to increase blood flow; and providing the therapeutic or diagnostic agent.
 17. The method of any of claims 14-16, further comprising chemically linking the modified C3 protein to an antibody with an affinity for a specific tissue prior to the delivering step.
 18. A method of treating a condition or disease associated with undesirable complement activation, comprising administering the modified C3 protein in an amount sufficient to deplete complement.
 19. The method of claim 18, wherein the condition or disease is selected from the group consisting of asthma, systemic lupus erythematosus, glomerulonephritis, rheumatoid arthritis, Alzheimer's disease, multiple sclerosis, myocardial ischemia, reperfusion, sepsis, hyperacute rejection, transplant rejection, cardiopulmonary bypass, myocardial infarction, angioplasty, nephritis, dermatomyositis, pemphigoid, spinal cord injury and Parkinson's disease
 20. A method of selecting a modified C3 protein, comprising: characterizing at least one property of the modified C3 protein to form a function profile of the modified protein; and matching the function profile with a disease or condition to be treated.
 21. A nucleic acid sequence encoding the modified C3 protein of claim
 1. 22. A composition comprising the modified human complement C3 protein of claim 1 and a pharmaceutically acceptable carrier.
 23. A expression system expressing the modified C3 protein of claim
 1. 